M Kulkarni, A Juvekar
anti-stress activity, anxiolytic activity, diazepam, tylophora indica
M Kulkarni, A Juvekar. Effect of roots of Tylophora indica (Burm.f.) on stress and anxiety in animal models. The Internet Journal of Pharmacology. 2009 Volume 8 Number 2.
The anti-stress activity of aqueous extract of
Stress is defined as the non-specific response of the body to any demand imposed upon it 1. It is known to alter the physiological homeostasis of the organism and complex mechanisms contribute to the breakdown in adaptational processes resulting in various visceral, endocrinal, and behavioural changes 2. Stress plays the main role in pathogenesis of mental disorders 3,4. A host of chronic psychiatric disease states like melancholic depression, anorexia nervosa, panic disorders, anxiety disorders and cognitive dysfunction have been reported to involve abnormality of stress axis 5. Anxiety disorders in particular, affect 1/8 th of total population worldwide and have become one of important research interest in psychopharmacology during this decade 6. The hallmark of anxiety disorders is marked, persistent and excessive or unreasonable fear that is experienced to a degree that significantly interferes with everyday life 7.
The prevention and management of these stress disorders remains a major clinical problem. Benzodiazepines (BDZs) appear to be effective against acute stress but fail to prevent the consequences of chronic stress. In addition, the problems of tolerance and physical dependence exhibited by BDZs on prolonged use, limit their utility 8. An answer to this vexing problem was first provided when Brekhman and Dardymov reported that some plant-derived agents could induce a state of non specific increase of resistance to affect internal homeostasis. Such agents, named adaptogen have been found to be effective in attenuating stress-induced adverse effects 9. Thus, in the current scenario, finding such adaptogenic agents that possess the ability to combat present day stress disorders along with possessing anxiolytic potential would offer added benefits in psychiatric treatment.
From the vast array of Materia Medica of the indigenous system so many plants have been reported to have activity against CNS disorders and thus act as very useful remedies for the alleviation of human suffering 10. Due to the chronic nature of psychiatric and neurological disorders, the therapeutic action of most psychotropic agents is dependent on long-term treatment and the consequent molecular and cellular adaptations that occur over time 11. In this context, administration of herbal plant-based formulation offers an added edge in treatment considering their cost effectiveness and the mild side effect profile compared to prescription alternatives.
Material and methods
Collection of plant material
The roots of
Preparation of the plant extract
Dried powdered roots were defatted with Pet ether (60-80°C) and successively extracted with water. The extract so obtained was dried using a vacuum evaporator (40°C) and used for further studies. The aqueous extract for oral administration was prepared in distilled water using 0.2% NaCMC as suspending agent.
Male Albino Wistar rats (150-200 gm body weight) and male Swiss Albino mice (20-25 gm body weight) were used for anti-stress and anxiolytic activities study respectively. The animals were housed in groups of five in clean polypropylene cage in standard laboratory conditions of temperature (25±2°C) with 12h/12h light and dark cycle. They had free access to food and water
Acute toxicity studies
Albino Wistar rats (150-200 gm body weight) and Swiss Albino mice (20-25 gm body weight) were used for acute oral toxicity study. The study was carried out as per the guidelines set by OECD and no adverse effects/mortality were detected in both rats and mice upto 5000 mg/kg p.o., during the 24-hr observation period. Based on the results obtained from this study, the doses for anti-stress and anxiolytic activity studies were narrowed down to 100 mg/kg, 250 mg/kg and 500 mg/kg for dose-dependent study.
Wistar rats were divided into six groups of six animals each. Group I animals served as vehicle control and received 0.2% NaCMC in distilled water. Group II animals received 0.2% NaCMC in distilled water and (stress); served as stress control. Group III animals were treated with diazepam (2 mg/kg
Chronic Cold Restraint Stress (CCRS) model:
After 21 days of pretreatment with vehicle/standard and test extracts to respective groups, same treatment was continued for seven more days and all animals except group I were subjected to chronic cold restraint stress during seven days. Rats were immobilized by placing them in plastic restraint containers which were placed in refrigerator (4°C for 1 hr) for seven continuous days. On the last day, the animals were treated as described earlier, exposed to cold restraint stress and sacrificed. Blood samples were collected to obtain plasma and adrenal gland and spleen were removed to record their weights 14,15.
Swiss Albino mice were divided into five groups of six each. Group I animals served as vehicle control (0.2% NaCMC). Group II animals were treated with diazepam (1 mg/kg
Elevated Plus maze Model (EPM)
The apparatus was a slightly modified version of the original plus-maze used for mice 16. The plus-maze elevated at 50 cm above a base, had four arms (6 cm wide, 16 cm long) extending from a central platform (8 X 8 cm). Two of the arms had side-walls (12 cm high), while the other two arms did not have side-walls. Mice were placed for period of 5 minutes in open arm and number of entries and time spent in open arm was noted 17.
The mice's light–dark box (30 X 30 X 10 cm) consisted of two parts, the light compartment and the dark-compartment with a volume ratio of 3:1. There was a hole (5 X 5 cm) in the bottom of the clapboard between the two compartments. A 60-W incandescent bulb above provided illumination of 700 lx for the open light-compartment and 0 lx for the enclosed dark compartment. At the start of the experiment, the mouse was placed in the middle of illuminated part of the cage. The number of crossings from dark to light compartment was registered during ten minutes 18.
The results obtained in the present study were expressed as the mean ± SD. The numerical results were evaluated by application of One-way Analysis of Variance (ANOVA) with post Bonferroni or Dunnett’s test wherever applicable for statistical significance. P < 0.05 was considered statistical significant.
Chronic cold restraint stress resulted in hypertrophy of adrenal gland and atrophy of spleen in stress control rats. Pretreatment with TI[AE]
Table 1: Effect of aqueous extract of
Values are mean ± S.D. (n = 6). Significantly different from vehicle control (#P < 0.001), significantly different from stress control group (aP < 0.05; bP < 0.01; cP < 0.001) by one way ANOVA followed by Post Bonferroni test.
Further, pretreatment with TI extract at all doses significantly (P < 0.05; P < 0.01; P < 0.001) restored back chronic cold restraint stress-induced alterations in plasma corticosterone, glucose, total protein, cholesterol and triglyceride levels (Table 2).
Table 2: Effect of pretreatment of aqueous extract of
Values are mean ± S.D. (n = 6). Significantly different from vehicle control (#P < 0.001), significantly different from stress control group (aP< 0.05, bP<0.01, cP< 0.001) by one way ANOVA followed by Post Bonferroni test.
Mice pretreated with TI[AE]
Table 3: Effect of aqueous extract of
Values are mean ± S.D. (n = 6). Significantly different from normal (aP < 0.05, bP < 0.01, cP < 0.001), by one-way ANOVA followed by Dunnett’s Test
Similarly, TI extract treated mice at all the doses showed significantly (P < 0.01) increased number of travellings from dark to light compartment compared to vehicle control mice (Table 4). The effect of test extract observed in both the models were comparable to standard anxiolytic, diazepam.
Table 4: Effect of aqueous extract of
Values are mean ± S.D. (n = 6). Significantly different from normal (aP< 0.05, bP<0.01, cP< 0.001), by one-way ANOVA followed by Dunnett’s Test
A wide variety of stresses, both physical and emotional, act via neural pathways to the hypothalamus to increase corticotropin-releasing hormone (CRH) secretion and hence, adrenocorticotropin hormone (ACTH) and cortisol secretion 19. Release of ACTH in stress stimulates adrenals to increase production of hormones epinephrine, norepinephrine, and corticosteroids 20. These hormones have profound effect on metabolic functions. Adrenaline raises blood sugar, while corticosteroid stimulates glycogenolysis and gluconeogenesis 21. Thus, increased cortisol influences mobilization of stored fat and carbohydrate reserve 22, which in turn increases blood glucose, protein, cholesterol, and triglyceride levels.
Chronic cold restraint stress (CCRS) model was used to deliberate the deleterious effect of chronic stress on the entire body system. Organ body weight indices of adrenal and spleen gland were used as vital markers for studying stress response, as involution of the spleen and increase in adrenal gland weight are primary consequences of chronic stress. The individual or combined weight of adrenal glands is likely to double under the influence of ACTH 23,24. Pretreatment with TI[AE] at all doses reversed increase in adrenal gland weight caused due to stress besides preventing spleen atrophy, thus inhibiting the basic signs of stress response. Likewise, test extract at all doses ameliorated the stress-induced rise in plasma corticosterone, glucose, cholesterol, triglyceride, and total protein levels in CCRS model. Thus, the mitigating effects shown by aqueous extract of
The two experimental models of anxiety, elevated plus maze and light and dark arena, are based on the assumption that unfamiliar, non-protective and brightly lit environmental stress provokes inhibition of normal behaviour. This normal behavioural inhibition is further augmented in the presence of fear or anxiety like state 25. In the elevated plus maze, the open arms are more fear provoking than the closed arms. It has been observed that anxiolytic drugs, diazepam-like, increase the number of arm entries and percentage of time spend in the open arms of an elevated plus maze and anxiogenic drugs have opposite effect 26. In the light and dark box paradigm, the brightly lit environment is a noxious environment stressor that inhibits the exploratory behaviour of rodents. Reduction in the number of entries and time spent in the light chamber are regarded as markers of anxiety 27.
Mice pretreated with TI extract at all the doses showed increase in number of entries and total time spent in open arm in elevated plus maze model. In addition, test extract treated group demonstrated increased number of travellings from dark chamber to light chamber in light-dark model. The behavioural changes in both the models indicative of decreased fear and decreased aversion to bright light, suggests the anxiolytic potency of the extract. The validity of both the anxiolytic models was supported by the observation that diazepam, a classical anxiolytic, showed preference to open arm and light chamber in elevated plus maze and light-dark model respectively.
In conclusion, the present study indicated that aqueous extract of roots of